CN107354127A - Effects of the LncRNA TUG1 in regulation and control PDLSCs Osteoblast Differentiations and regeneration - Google Patents

Effects of the LncRNA TUG1 in regulation and control PDLSCs Osteoblast Differentiations and regeneration Download PDF

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CN107354127A
CN107354127A CN201710561399.4A CN201710561399A CN107354127A CN 107354127 A CN107354127 A CN 107354127A CN 201710561399 A CN201710561399 A CN 201710561399A CN 107354127 A CN107354127 A CN 107354127A
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tug1
lncrna
stem cell
regeneration
periodontal ligament
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CN107354127B (en
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魏福兰
王春玲
何琴
谷秀格
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Shandong University
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Shandong University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources

Abstract

The invention discloses effects of the LncRNA TUG1 in regulation and control PDLSCs Osteoblast Differentiations and regeneration.The present invention regulates and controls outer inside PDLSCs Osteoblast Differentiations and regeneration test by studying LncRNA TUG1, LncRNA TUG1 have played important regulating and controlling effect in periodontal ligament stem cell Osteoblast Differentiation and tissue regeneration processes, LncRNA TUG1 can be as the potential target spot of tissue regeneration therapies, promotion is developed new on LncRNA TUG1 related drugs or the factor, and most it applies to regenerative medicine field at last, reach clinically really precisely medical treatment.

Description

Effects of the LncRNA-TUG1 in regulation and control PDLSCs Osteoblast Differentiations and regeneration
Technical field
The present invention relates to oral tissue regeneration domain technology field, and in particular to LncRNA-TUG1 regulation and control PDLSCs into Effect in bone differentiation and regeneration.
Background technology
Oral cavity stem-cell research realizes that biology regeneration and biological function reparation provide for human teeth and periodontium May.In recent years, new research is brought to biology regeneration on the progress of tooth steam cell and tissue engineering technique Direction.Tooth steam cell can use soft or hard group with human body multiple location with advantages such as energetic, convenient material drawing, low immunological rejections Knit regeneration, reparation and regeneration such as tooth and periodontium, repaired after the wound of eyes, the healing of skin and regeneration, fat it is thin The tissue reconstruction of born of the same parents, self-regeneration of immune system etc..Periodontal ligament stem cell (PDLSCs) is a kind of in clinical oral work It is easier to the adult stem cell obtained；A kind of and adult stem cell with multi-lineage potential and self-renewal capacity.From 04 Year Seo etc. it is successfully separated and identifies from the periodontal membrane tissue of human body using single cell clone technology, periodontal ligament stem cell is with regard to standby Paid close attention to by dentistry and other biological regeneration field researcher.It can form bone, soft by appropriate induction in vitro The Various Tissues such as bone, nerve, fat, blood vessel；Possess the potential ability for being divided into Gegenbaur's cell or cementoblast, and can be Cementum-parodontium-alveolar bone spline structure thing is formed after transplanting in vivo, is bone tissue maintenance, osteanagenesis, the reparation of clinical oral And the periodontal remodeling of correction provides important clinical value.
Present inventor seminar takes the lead in proposing to carry out biological tooth using tooth related stem cells early stage in the world The new concept of root regeneration, and carried out the inside and outside experiment of serial dependent body, go out can be just for successful regeneration such as on miniature pig animal model The biological root of the tooth often to function；The periodontal ligament stem cell diaphragm that autologous allosome periodontal ligament stem cell and Vc induce is used for tissue Regeneration, achieves good effect；Utilize cryogenic freezing technology, the biology performance and storage of optimization periodontal ligament stem cell diaphragm Condition, carry out the autologous regeneration of allosome stem cell tissue and reparation for different time points and provide possibility.
In human genome, there are about 93% DNA can all be transcribed into RNA, but only 2% RNA has encoding proteins The function of matter, numerous DNA are finally transcribed into non-coding RNA.Long-chain non-coding RNA (long non coding RNA, LncRNA) it is non-coding RNA of a kind of length more than 200 nucleotides.It is considered as the noise in transcription in the past, does not have Practical function.But in the last few years, goed deep into research, scientists find it in epigenetic, transcription and posttranscriptional gene Important regulating and controlling effect is all played in expression.Studies have shown that LncRNAs participates in the adjustment effect of a variety of vital movements：Can be to egg White encoding gene carries out epigenetics regulation；Direct regulatory gene transcription and protein degradation；Participate in the life of some organelles Transported into the subcellular fraction of process and intracellular molecules；It is and often closely related with gene imprinting phenomenon.At present, LncRNAs is studied most Important effect may be by number of mechanisms and promote or suppress the occurrence and development of tumour and attack transfer, including hepatocellular carcinoma, Cancer of pancreas, leukaemia etc..In terms of stem-cell research, research in recent years shows some LncRNA and the biological behaviour of stem cell It is closely related.In human adipose's stem cell skeletonization into atomization, LncRNA-MEG3 is by regulating and controlling miR-140-5p so as to reaching Strengthen to the fat for suppressing ADMSC to differentiation and induced osteogenesis to ability to express；Equally, reduce in fat stem cell LncRNA-MIAT expression, its differentiation to Gegenbaur's cell can be promoted.In human marrow mescenchymal stem cell, knock out The expression of skeletonization differentiation gene can be accelerated after Long Non-Coding RNA NONHSAT009968；LncRNA-H19 is participated in Differentiation of the bone marrow stroma stem cell to Gegenbaur's cell and nerve cell, when under H19 timing BMSC can differentiating into nerve cells energy Power strengthens；Meanwhile lncRNA (Esco2, Pcdhb18 and RGD1560277) also assist in regulation and control BMSC it is thin to nerve cell and skeletonization Born of the same parents break up.
Taurine up-regulated gene 1 (Taurine upregulated gene 1, TUG1) is located at human chromosomal 22q12.2, length are about 7.1kb, have several open reading frames but most long coding is no more than 100 amino acid, therefore It is a kind of long-chain non-coding RNA to think it.TUG1 is found in mouse retinal cells earliest, retina cell nucleus and There is expression in cytoplasm；Retina cell's decreased growth, apoptosis capacity increase can be caused after TUG1 is knocked out, cause mouse to send out Educate deformity.LncRNA-TUG1 can recruit polycomb repressive complex 2 (PRC2) albumen to methylate, from And regulate and control the expression of some cell growth control genes.It is several for three days after retinal progenitor cells progress TUG1siRNA transfections Related to Apoptosis path gene expression up-regulation (such as Klf2), cell death increase, TUG1 normal expression may be for Suppress some gene participating in apoptosis to play an important role.TUG1 has adjustment effect to SM22 α in vascular smooth muscle cells. After silence TUG1, cell growth slows down or apoptosis increase, SM22 α mRNA up-regulated expressions；It can suppress compound with more comb albumen EZH2 protein bindings in body 2 (PRC2), so as to regulate and control the expression of some growth control genes；There is EZH2 in endochylema methyl to turn Move in enzymatic activity and the forming process of the cell pseudopodium in PDGF inductions and play a crucial role, draw TUG1 and EZH2, F-actin Between interaction mechanism and its adjustment effect to vascular smooth muscle cells skeleton.Researcher also found TUG1 promoters Nearby include some highly conserved P53 bindings sites, simultaneously up-regulated expression can be stimulated by the P53 DNA damage responses mediated. In addition, after TUG1 is combined with PRC2, then combined with target gene and carry out chromatin modification, play controlling gene expression function and Realize the expressional function of silence target gene.TUG1 is a kind of regulatory factor, although its not encoding proteins matter, can directly with RNA form by with a variety of transcription factor interactions, regulate and control polygenic expression, for mankind's normal growth and development process Play an important roll.But effects of the LncRNA-TUG1 in periodontal ligament stem cell and with periodontal ligament stem cell Osteoblast Differentiation and Regeneration dependent interaction is studied, and has no report at present.
The content of the invention
For above-mentioned prior art, inventor combine previous experiments basis (grant of national natural science foundation problem, No.81200758), regulate and control outer experiment inside PDLSCs Osteoblast Differentiations and regeneration by studying LncRNA-TUG1, carry out Molecular level study and tissue regeneration in vivo research, as a result show, LncRNA-TUG1 is in periodontal ligament stem cell Osteoblast Differentiation and group Knit and important regulating and controlling effect played in regenerative process, by reversely proving, when LncRNA-TUG1 lower, PDLSCs skeletonization to point Change and paradenlal tissue regeneration ability is suppressed, LncRNA- in the regeneration of periodontal ligament stem cell mediating tissue is illustrated by the present invention TUG1 adjusting function, oral tissue regeneration is promoted to provide important foundation for regulation and control tooth steam cell.
Specifically, the present invention relates to following technical scheme：
First, an object of the present invention be to provide LncRNA-TUG1 as target spot regulation and control periodontal ligament stem cell into Application in bone differentiation and/regeneration.
Specifically, the regulation and control include positive regulation and control and/negative regulation, positive regulation and control refer to promote periodontal ligament stem cell Osteoblast Differentiation With/regeneration, negative regulation refers to suppress periodontal ligament stem cell Osteoblast Differentiation and/regeneration.
LncRNA-TUG1 expression improves, and can promote periodontal ligament stem cell Osteoblast Differentiation and/regeneration；LncRNA- TUG1 expression declines, and PDLSCs skeletonization is suppressed to differentiation and paradenlal tissue regeneration ability.
Based on above-mentioned application, the invention discloses a kind of LncRNA-TUG1 accelerator to prepare periodontal ligament stem cell skeletonization Purposes in differentiation and/regeneration medicine.
The LncRNA-TUG1 accelerator refers to any activity that can improve LncRNA-TUG1, increase LncRNA- The material of TUG1 expression quantity, such as the LncRNA-TUG1 of increase LncRNA-TUG1 expression quantity over-express vector.
The invention also discloses LncRNA-TUG1 inhibitor to prepare suppression periodontal ligament stem cell Osteoblast Differentiation and/tissue Purposes in regenerating medicine.
In above-mentioned application, the LncRNA-TUG1 inhibitor includes antagonist, lower adjustment, retarding agent, blocking agent etc.. Any activity that can reduce LncRNA-TUG1, the material of reduction LncRNA-TUG1 expression quantity are used equally for the present invention.
In preferred embodiment, the LncRNA-TUG1 inhibitor carries to suppress the slow virus of LncRNA-TUG1 expression Body.
Based on above-mentioned application, the invention discloses a kind of side for promoting periodontal ligament stem cell Osteoblast Differentiation and/regeneration Method, methods described include the activity for improving LncRNA-TUG1 and/increase LncRNA-TUG1 expression.
In preferred embodiment, methods described also includes that LncRNA-TUG1 activity and/increase LncRNA- will be improved Periodontal ligament stem cell after TUG1 expression, form periodontal ligament stem cell compound with artificial bone supporting material and prepare osteogenic materials Step.
The invention also discloses a kind of method for suppressing periodontal ligament stem cell Osteoblast Differentiation and/regeneration, methods described Expression including suppressing LncRNA-TUG1.
Periodontal ligament stem cell has a variety of differentiation potentials, can form bone, cartilage, god by appropriate induction in vitro Through, Various Tissues such as fat, blood vessel, (even if during directional induction) periodontal ligament stem cell also can be with a variety of in induction The generation (Gegenbaur's cell+adipocyte, Gegenbaur's cell+vascular cell etc.) of noble cells, Osteoblast Differentiation is suppressed by part, can Effectively to induce stem cell to other particular organization's direction differentiation.
Secondly, the second object of the present invention is the Osteoblast Differentiation for providing a kind of regulation and control periodontal ligament stem cell and/tissue again Raw pharmaceutical composition, described pharmaceutical composition contain above-mentioned LncRNA-TUG1 accelerator or the LncRNA-TUG1 suppressions of effective dose Preparation.
Further, also included in described pharmaceutical composition：Pharmaceutically acceptable carrier.
" pharmaceutically acceptable carrier " is conventional drug administration carrier, including various excipient or diluent etc..
The present invention achieves following beneficial effect：
The present invention regulates and controls outer inside PDLSCs Osteoblast Differentiations and regeneration test by LncRNA-TUG1, it was confirmed that LncRNA-TUG1 has played important regulating and controlling effect in periodontal ligament stem cell Osteoblast Differentiation and/tissue regeneration processes； LncRNA-TUG1 can be as the potential target spot of tissue regeneration therapies, and promotion is developed new on LncRNA-TUG1 related drugs Or the factor, and most it applies to regenerative medicine field at last, reaches clinically really precisely medical treatment.
Brief description of the drawings
The Figure of description for forming the part of the application is used for providing further understanding of the present application, and the application's shows Meaning property embodiment and its illustrate be used for explain the application, do not form the improper restriction to the application.
Fig. 1 periodontal ligament stem cell biological characteristics experimental results, it is primary that PDLSCS stem cells are separately cultured situation (A) display It is commissioned to train foster cell (scale bar with the 3rd：200 μm), the ALP dyeing of Multidirectional Differentiation ability measure, Alizarin red staining (B), skeletonization Correlation factor determines (C)
Fig. 2 periodontal ligament stem cells skeletonization to the situation of change after induction, A. periodontal ligament stem cells skeletonization to after induction 7d, Obvious LncRNA is expressed in RT-qPCR screenings；B. periodontal ligament stem cell skeletonization changes to induction different time, TUG1 expression
Fig. 3 transfection slow virus PDLSCs immunofluorescence situation of change, A. transfect the slow virus carrier of disturbance target spot, PDLSCs shows fluorescent microscopy images Scale bar：200um.；B.RT-qPCR checkings, disturbance target spot silence TUG1 efficiency feelings Condition
Fig. 4 Periodontal ligament stem cells skeletonization is to induction different time, correlation skeletonization index situation of change during silence TUG1, A.ALP dyes (1w) (scale bar：200μm)；B. Alizarin red staining (3w) (scale bar：200μm)；C.ALP activity；D. Calcium ion concentration；E. the expression of osteogenic factor (ALP, Runx2, OCN), as a result show silence TUG1 groups compared with empty carrier group and Blank control group is in reducing tendency, and empty carrier group is not significantly different (with * p between the two with non-adding carrier induction group<0.05 has system Meter learns meaning)
Fig. 5 LncRNA-TUG1 regulate and control periodontal ligament stem cell tissue regeneration in vivo ability.HE dyes (8w, 12w), scale bar：100μm
Embodiment
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag Include " when, it indicates existing characteristics, step, operation and/or combinations thereof.
In order that technical scheme can clearly be understood by obtaining those skilled in the art, below with reference to tool The embodiment of body describes technical scheme in detail.
The periodontal ligament stem cell biological characteristics of embodiment 1
1st, the separation and culture of Periodontal ligament stem cell
Sterile first and second premolar teeth for pulling out people's impacted third molar or orthodontic therapy, gently peels off its week under anesthesia The periodontium enclosed, take the periodontium in stage casing, cleaned, shredded repeatedly with PBS, be placed in enzyme containing NTx (3g/L) and The digestive juice of the enzymes of Dispase II (4g/L), digest 40 minutes at 37 DEG C, cross 70um cell sieves and collect cell, 1000 revs/min, Centrifugation 5 minutes, removes the liquid on upper strata, single cell suspension is suspended into again with fresh nutrient solution.Cell is inoculated in 25cm2 In Tissue Culture Flask, (contain 20% hyclone, 2mmol/L glutamine, 100U/ml penicillin and 100 μ in α-MEM culture mediums G/ml streptomysins) in 37 DEG C, 5%CO2Culture, every 2-3 days, changes nutrient solution 1 time, and cell life is observed under inverted microscope Long situation.When cell growth to 80% converging state, 1 is pressed with 0.25% trypsase：2 or 1：3 had digestive transfer cultures.(take the 3rd Cell completes subsequent experimental from generation to generation)
2nd, the expression of periodontal ligament stem cell surface marker
Using flow cytometry and immunofluorescence art, observe between periodontal ligament stem cell STRO-1, CD146, CD45, CD31 etc. The expression of mesenchymal stem cells mark.
3rd, Multidirectional Differentiation ability
By periodontal ligament stem cell respectively in skeletonization to inducing culture and fat to inducing culture culture 4 weeks, Ran Houfen Its multi-lineage potential is not detected by Alizarin red staining, oil red O stain and RT-PCR technology.
Experimental result is as shown in figure 1, it can be seen from the results that the PDLSCs of the invention being separately cultured is by identifying and luring Lead, Gegenbaur's cell can be divided into, the multi-lineage potential for possessing stem cell.
Embodiment 2 screens LncRNA-TUG1, and cellular localization and the expression change in skeletonization into induction are carried out to it 1st, periodontal ligament stem cell is distinguished into skeletonization to induction 0d, 7d, RNA is extracted, by RT-qPCR, it is determined that it is obvious to filter out expression Purpose non-coding RNA, and carry out variation tendency of the different time skeletonization to induction..
Experimental result is as shown in Fig. 2 filter out the obvious LncRNA-TUG1 of expression, and tentative confirmation TUG1 is in PDLSCs Expression trend in Osteoblast Differentiation.
2nd, slow virus carrier is built
According to people's LncRNA-TUG1 sequences (ID:55000) primer is designed, primer is by the limited public affairs of Shanghai neck section biotechnology Department synthesizes and purified.The pGenesil-1 plasmids of linearisation are connected to after annealing, structure LncRNA-TUG1 (silence) expression carries Body slow virus, suppress LncRNA-TUG1 expression (because TUG1 sequences are excessive, it is extremely difficult to successfully build over-express vector, therefore this hair The carrier that bright structure suppresses LncRNA-TUG1 expression carries out reversely research demonstration).
Building process used carrier and primer are as follows：
Vector：pLenO-GFP
Lenti-hTUG1-NC sequences：TTCTCCGAACGTGTCACGT(SEQ ID NO:1)(Negative Control)
Lenti-hTUG1-sh1
GPH-hTUG1-SH1 Forward Sequence
5’GATCCCTGTTGACCTTGCTGTGAGAACTTCCTGTCAGATTCTCACAGCAAGGTCAACAGTTTTTG3’ (SEQ ID NO:2)
GPH-hTUG1-SH1 Reverse Sequence
5’AATTCAAAAACTGTTGACCTTGCTGTGAGAATCTGACAGGAAGTTCTCACAGCAAGGTCAACAGG3’ (SEQ ID NO:3)
Lenti-h TUG1-sh2
GPH-hTUG1-SH2 Forward Sequence
5’GATCCGCTTGGCTTCTATTCTGAATCCTTTCTTCCTGTCAGAAAAGGATTCAGAATAGAAGCCAAGC TTTTTG3’(SEQ ID NO:4)
GPH-hTUG1-SH2 Reverse Sequence
5’AATTCAAAAAGCTTGGCTTCTATTCTGAATCCTTTTCTGACAGGAAGAAAGGATTCAGAATAGAAGC CAAGCG3’(SEQ ID NO:5)
Lenti-h TUG1-sh3
GPH-hTUG1-SH3 Forward Sequence
5’GATCCGCTACAACTATCTTCCTTTACCTTCCTGTCAGAGTAAAGGAAGATAGTTGTAGCTTTTTG 3’(SEQ ID NO:6)
GPH-hTUG1-SH3 Reverse Sequence
5’AATTCAAAAAGCTACAACTATCTTCCTTTACTCTGACAGGAAGGTAAAGGAAGATAGTTGTAGCG 3’(SEQ ID NO:7)
Lenti-h TUG1-sh4
GPH-hTUG1-SH4 Forward Sequence
5’GATCCGCAAGAGATAACTATGAAAGCCTTCCTGTCAGAGCTTTCATAGTTATCTCTTGCTTTTTG 3’(SEQ ID NO:8)
GPH-hTUG1-SH4 Reverse Sequence
5’AATTCAAAAAGCAAGAGATAACTATGAAAGCTCTGACAGGAAGGCTTTCATAGTTATCTCTTGCG 3’(SEQ ID NO:9)
Cell is grouped：Control groups, sh-NC groups, sh-TUG1-1# groups, sh-TUG1-2# groups, sh-TUG1-3# Group, sh-TUG1-4# groups, virus is separately added into according to the requirement of slow-virus transfection step specification.Normal incubation medium is gained after 12h, In fluorescence microscopy Microscopic observation after incubation 24h, there is green fluorescence explanation and transfect successfully.The screening transfection stable time (24h, 48h, 72h, 96h), it is 72h finally to select transfection time, and MOI values are 20.Total serum IgE is extracted, completes follow-up RT-qPCR correlation Checking.
Screening verification result is shown：The silencing efficiency of sh-TUG1-2# groups is most obvious (as shown in Figure 3).
3rd, by periodontal ligament stem cell distinguish skeletonization to induction different time (0,1,3,7,10,14d), LncRNA-TUG1's Expression change
Cell is cleaned with serum free medium, sh-NC and sh-TUG1-2# slow virus adds according to transfection procedure specification Enter into cell.Use normal culture medium after 12h instead, be incubated 72h, skeletonization is given after stabilization to be transfected and is trained to inducing culture Support.Complete functional studies of the follow-up LncRNA-TUG1 to periodontal ligament stem cell Osteoblast Differentiation.
Experimental result is as shown in Figure 4, the results showed that, LncRNA-TUG1 in PDLSCs skeletonization to induction different time, it is overall Expression trend is in rising trend；ALP is dyed and Alizarin red staining shows that PDLSCs skeletonization has substantially to induction with blank control group Difference；The expression of ALP activity and osteogenic ability correlation factor (ALP, Runx2, OCN) raises compared with the expression of blank control group, Demonstrate again that PDLSCs possesses ability of the skeletonization to cell differentiation.
The LncRNA-TUG1 of embodiment 3 regulates and controls the research of periodontal ligament stem cell tissue regeneration in vivo
Periodontal ligament stem cell compound is implanted to nude mice dorsal sc, it is observed and organizes the formation of ability.
1st, slow virus sh-NC empty carriers and sh-TUG1-2# are transfected into periodontal ligament stem cell respectively
Using tissue block method and the periodontal ligament stem cell of enzyme digestion culture people, eugonic third generation parodontium is done Cell is seeded in 60mm culture dishes, be trained be divided into α-MEM culture mediums (contain 15% hyclone, 2mmol/L glutamine, 100U/ml penicillin, 100 μ g/ml streptomysins).Slow virus sh-NC empty carriers and sh-TUG1-2# transfection parodontium are done respectively Cell, experiment are divided into 3 groups：(1) normally culture periodontal ligament stem cell group (2) sh-NC empty carriers transfect periodontal ligament stem cell group (3) Sh-TUG1-2# transfects periodontal ligament stem cell group
2nd, the preparation of biologic bracket material
Periodontal ligament stem cell by autoclaved β-TCP biologic bracket materials and in vitro culture is co-cultured, Ratio 50mg：1*107Individual cell.It is randomly divided into 3 groups：(1) normally culture periodontal ligament stem cell film+β-TCP groups (2) sh-NC is empty Carrier transfection periodontal ligament stem cell+β-TCP groups (3) sh-TUG1-2# transfection periodontal ligament stem cell+β-TCP groups
3rd, return and plant in timbering material/stem cell complex body
Nude mice is randomly divided into 3 groups：(1) normally culture periodontal ligament stem cell film+β-TCP groups (2) sh-NC empty carriers transfect tooth After all film stem cell+β-TCP groups (3) sh-TUG1-2# transfections periodontal ligament stem cell+β-TCP organize back plant 8w, 12w, put to death respectively Nude mice.
A. imageological examination：X-ray and CT examination, tissues observed regeneration situation.
B. histological observation：Put to death animal and obtain sample, fixed, decalcification, carry out HE dyeing, observation biological support tissue is again Raw situation.
Experimental result is shown, lowers obvious (the H＆E dyeing of change decline of tissue regeneration in vivo ability after LncRNA-TUG1 After showing mineralising induction 8w, 12w, it was demonstrated that LncRNA-TUG1 has facilitation to tissue regeneration ability).
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Shandong University
<120>Effects of the LncRNA-TUG1 in regulation and control PDLSCs Osteoblast Differentiations and regeneration
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Claims (10)

  1. Applications of the 1.LncRNA-TUG1 as target spot in regulation and control periodontal ligament stem cell Osteoblast Differentiation and/regeneration, its feature It is, in the application, LncRNA-TUG1 expression improves, and promotes periodontal ligament stem cell Osteoblast Differentiation and/regeneration； LncRNA-TUG1 expression declines, and periodontal ligament stem cell skeletonization is suppressed to differentiation and paradenlal tissue regeneration ability.
  2. Purposes of the 2.LncRNA-TUG1 accelerator in periodontal ligament stem cell Osteoblast Differentiation and/regeneration medicine is prepared.
  3. 3. purposes according to claim 2, it is characterised in that the LncRNA-TUG1 accelerator refers to improve LncRNA- TUG1 activity, the material for increasing LncRNA-TUG1 expression quantity.
  4. 4.LncRNA-TUG1 inhibitor is preparing the purposes in suppressing periodontal ligament stem cell Osteoblast Differentiation and/regeneration medicine.
  5. 5. purposes according to claim 4, it is characterised in that the LncRNA-TUG1 inhibitor includes but is not limited to drop Antagonist, lower adjustment, retarding agent, the blocking agent of low LncRNA-TUG1 activity and/reduction LncRNA-TUG1 expression quantity, preferably , the slow virus carrier that the LncRNA-TUG1 inhibitor is expressed for suppression LncRNA-TUG1.
  6. 6. a kind of method for promoting periodontal ligament stem cell Osteoblast Differentiation and/regeneration, methods described includes improving LncRNA- The expression of TUG1 activity and/increase LncRNA-TUG1.
  7. 7. according to the method for claim 6, it is characterised in that methods described also includes that LncRNA-TUG1 work will be improved Property and/increase LncRNA-TUG1 expression after periodontal ligament stem cell, with artificial bone supporting material formed periodontal ligament stem cell it is compound Thing prepares the step of osteogenic materials.
  8. 8. a kind of method for suppressing periodontal ligament stem cell Osteoblast Differentiation and/regeneration, methods described includes suppressing LncRNA- TUG1 expression.
  9. 9. a kind of Osteoblast Differentiation of regulation and control periodontal ligament stem cell and the pharmaceutical composition of/regeneration, described pharmaceutical composition contain There are LncRNA-TUG1 accelerator or the LncRNA-TUG1 inhibitor of effective dose.
  10. 10. pharmaceutical composition according to claim 9, it is characterised in that also included in described pharmaceutical composition：Pharmaceutically Acceptable carrier.
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CN110616190A (en) * 2019-02-19 2019-12-27 中国人民解放军第四军医大学 Method for regulating and controlling osteogenic differentiation of periodontal ligament stem cells based on extracellular matrix
CN110616190B (en) * 2019-02-19 2023-08-18 中国人民解放军第四军医大学 Method for regulating and controlling periodontal ligament stem cell osteogenic differentiation based on extracellular matrix
CN110241196A (en) * 2019-05-10 2019-09-17 山东大学 Application of the circRNA PRKD3 in periodontal ligament stem cell Osteoblast Differentiation
CN110241196B (en) * 2019-05-10 2020-07-28 山东大学 Application of circRNA PRKD3 in osteogenic differentiation of periodontal ligament stem cells
CN110305867A (en) * 2019-07-10 2019-10-08 山东如戴生物科技有限公司 Regulate and control method and its reagent that fat stem cell stemness maintains ability
CN112239744A (en) * 2020-10-20 2021-01-19 青岛市妇女儿童医院（青岛市妇幼保健院、青岛市残疾儿童医疗康复中心、青岛市新生儿疾病筛查中心） Interfering RNA capable of promoting osteogenic transformation of dental pulp stem cells and application thereof
CN112342217A (en) * 2020-11-11 2021-02-09 杨桂梅 Periodontal ligament stem cell proliferation and osteogenic differentiation promoter
CN112342217B (en) * 2020-11-11 2021-04-16 杨桂梅 Periodontal ligament stem cell proliferation and osteogenic differentiation promoter
CN113413489A (en) * 2021-06-23 2021-09-21 重庆医科大学附属口腔医院 Let-7 a-containing periodontal defect repair system
CN113413489B (en) * 2021-06-23 2022-06-10 重庆医科大学附属口腔医院 Let-7 a-containing periodontal defect repair system

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